The group of complex terpene-type compounds known as taxanes have proven to have important anti-cancer properties. The reference herein to taxane, taxane-type compounds or taxol will be shorthand for this recognized group of compounds having anti-cancer activity and other properties.
The primary source of the anti-cancer drug taxol is bark tissue of Taxus brevifolia, the Western (Pacific) yew. The supply of this drug is limited by the number of these slow-growing trees available for harvest. Using current methods, trees are cut and bark is removed. The most bark is, of course, obtained from mature trees. Cutting, however, kills the tree and reharvesting awaits 20 years or more for regrowth. The rate of cutting threatens wild trees with extinction.
The yield of taxol/taxane compounds is measured in micrograms per pound of plant bark where the highest concentration of taxol is normally found. For example, to obtain 1 gram of taxol requires harvesting some 22 lbs. of bark which represents 2-3 mature trees of age 20-40 years. A typical cancer treatment involves from 1 to 2 grams per patient over the course of the treatment regime extending 3-6 months.
A variety of processes have been proposed for sources of taxol/taxanes and their derivatives, including: Callus cell culture techniques, managed tree farms, and classic organic synthesis.
For example, U.S. Pat. No. 5,019,504 of Christen et al. is directed to culturing selected tissue, preferably bark or cambial tissue, from Taxus brevifolia. Since cultured plant tissue ordinarily does not produce the same compounds in culture, culturing is not always successful and is tricky to maintain. That is, taxol production is non-growth associated, involving secondary metabolism. Culturing may interfere with the secondary metabolism products, although there is no uncoupling of the post-growth relationship as taxol/taxanes are not usually produced during the initial callus proliferation stage. Further, taxol production after suspension cell culture growth is best induced by fungal or inorganic elicitors in subcultures, the best results being shown in subcultures supported on special agar media.
In Science News, Feb. 22, 1992, page 124, it is reported that a variety of workers are attempting various organic syntheses, which are difficult. Growing vast numbers of trees on managed tree farms is another approach, but that takes time as taxol is produced in the older plants. It is climate limited, labor intensive, and requires an enormous investment in land and harvesting equipment.
Gasser and Fraley, in an article entitled Transgenic Crops, Scientific American, June 1992, p. 62 describes the formation of crown galls by the plant pathogen Agrobacterium tumefaciens. "This bacterium can transfer a portion of its DNA into plant cells. It does so by introducing a set of genes into one or more of its own [sic] DNA fragments. These fragments called transferred DNA (T-DNA), then integrate into chromosomes of infected plant cells and induce the cells to produce elevated levels of plant hormones. These hormones cause the plant to form novel structures, such as tumors or prolific root masses, that provide a suitable environment and nutrient source for the Agrobacterium strain. This bacterial infection is crown gall disease." The Sanford DNA particle gun used for plant genetic transformation is shown in this article.
Also, De Cleene, in a letter in Speculations in Science and Technology, Vol. 3, No. 3, August 1980, Elsevier Sequoia, Lausanne, pp. 353-356, proposed consideration of in vitro cell and tissue cultures of crown gall tissue as a source of physiologically active substances because of their possible greater metabolic activity. He cautioned that "It is true that the biosynthetic pathways are not necessarily the same for normal and crown gall tissues. Biochemical differences between both tissue types have been suggested in the literature, but crown gall tumors nevertheless seem to reflect the physiological state of the host plant from which it developed. So there is a good chance that therapeutically active compounds are synthersized in both healthy and crown gall tumors of a certain plant species." In inviting experimentation along this line of speculation, De Cleene listed some 22 species, one of which was "Taxus brevifolia Nutt. (Western yew): anti-tumor agent, cyanogenetic glycoside (taxiphyllen)."
Subsequent research dampened De Cleene's speculations. Thus, Norton et al. in Phytochemistry Vol. 24, No. 4 pp. 719-722 (1985) measured thiophene concentrations in crown gall tissue produced by Agrobacterium tumefaciens strains A208 and A277 of Tagetes patula plants (Marigold-French Dwarf Double, Sparky Mixed) and concluded that: "The results showed it is not possible to predict the amounts of secondary metabolites produced as a result of transfers of genetic material from infected plants to crown galls and then to transformed callus tissues." They also noted that: "Most of the publications to date (1985) contain results which show a reduced yield of secondary metabolite production by crown gall tissues or transformed callus tissues."
Huizing, H., Hibma, J. and Wichers, H., reported in Acta Botanica Neerlandica, 35(1):47 (1986) on their investigation of the feasibility of production of L-DOPA on crown-gall tumour tissue of Nucuna puriens (a dicot). Tumors were initiated on plants, removed and placed in culture to obtain hormone autotrophic cell lines. Transformed cells did not produce more L-DOPA than did cells which were grown in the presence of growth regulators.
Luckner, M., Moldenhauer, E., First, B., and Diettrich, B., in Plant Medica 56:498-499 (1990) reported on cardenolide accumulation in crown galls and cell lines obtained by transformation of Digitalis lanata (a dicot) with wild-type strains of Agrobacterium tumefaciens. Tumors were induced on sterile leaf disks obtained from greenhouse or in vitro plants and the tissue analyzed for digitoxin derivatives. The average content of products was only 30% that of the leaves the galls were attached to, but in some cases exceeded concentrations in the leaf. For digoxin, the results were different. Crown galls only contained about 1% that of the leaf.
Cosio, E., Norton, R., Towers, E., Finlayson, A., Rodriguez, E., and Towers, G. reported in J Plant Physiol 124:155-164 (1986) on production of antibiotic thiarubrines by a crown gall tumor line of Chaenactis douglasii (a dicot). They worked with a crown gall tumor line in vitro. They interpret their results as evidence of tissue organization in tumors being necessary for product formation. No data on freshly excised tumors was presented.
Accordingly, there is a need in the art to provide a process for increasing the rate of growth and concentrations in naturally growing Taxus tissue for increased yields of taxol/taxane-compounds.